Article

Modulation of Gene Expression in a Sterile Atopic DermatitisModel and Inhibition of Staphylococcus aureus Adhesionby Fucoidan

Ah Young Park 1,* , Maureen Bourtembourg 2, Aline Chrétien 2, Roland Hubaux 2 , Céline Lancelot 2,Michel Salmon 2 and J. Helen Fitton 1,*

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Citation: Park, A.Y.; Bourtembourg,

M.; Chrétien, A.; Hubaux, R.;

Lancelot, C.; Salmon, M.; Fitton, J.H.

Modulation of Gene Expression in a

Sterile Atopic Dermatitis Model and

Inhibition of Staphylococcus aureus

Adhesion by Fucoidan.

Dermatopathology 2021, 8, 69–83.

https://doi.org/10.3390/

dermatopathology8020012

Academic Editor: Gürkan Kaya

Received: 3 March 2021

Accepted: 18 March 2021

Published: 25 March 2021

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4.0/).

1 Marinova Pty Ltd., 249 Kennedy Drive, Cambridge TAS 7170, Australia2 StratiCELL, Crealys Science Park, rue Jean Sonet 10, B-5032 Isnes, Belgium;

mbourtembourg@straticell.com (M.B.); achretien@straticell.com (A.C.); rhubaux@straticell.com (R.H.);clancelot@straticell.com (C.L.); msalmon@straticell.com (M.S.)

* Correspondence: ahyoung.park@marinova.com.au (A.Y.P.); helen.fitton@marinova.com.au (J.H.F.)

Abstract: Atopic dermatitis is a multifactorial pathology that includes perturbations of gene ex-pression and increased adhesion of Staphylococcus aureus. Fucoidans are seaweed-derived sulfatedfucose-rich polysaccharides that are known to be anti-inflammatory and may inhibit adhesion ofpathogens. Fucoidan was assessed for effects on gene expression of an in vitro 3D model of atopicdermatitis. It was also assessed for inhibitory effects on the adhesion of bacteria onto 3D reconstructedskin. Fucoidan significantly altered gene expression in the atopic dermatitis model, and there wasa trend to reduce periostin levels. Fucoidan significantly inhibited the adhesion of Staphylococcusaureus and Cutibacterium acnes but did not affect the adhesion of Staphylococcus epidermidis. Fucoidanmay be a useful topical agent to assist in the management of atopic dermatitis.

Keywords: atopic dermatitis; Staphylococcus aureus; fucoidan; periostin; reconstructed human epidermis

1. Introduction

Atopic dermatitis (AD) is a multifactorial debilitating skin condition that is associatedwith the bacteria Staphylococcus aureus, and alterations in the expression of genes involvedin barrier function, itch and inflammation. A combination of environmental exposure,epidermal barrier disruption and immune dysregulation are all part of this condition,which includes severe pruritus and eczematous changes. Bacterial involvement may notbe an initiator of the condition, but a sequel to it. Treatment of atopic dermatitis mayinvolve both topical and systemic steroids, methotrexate, biological immunomodulatorsand antibiotics. The disease burden of AD is considerable, affecting people of all ages andethnicities [1], and there is an unmet need for effective, non-toxic treatments.

Fucoidans are brown seaweed-derived sulfated fucose-rich polysaccharides that areknown to be anti-inflammatory [2,3] and may inhibit the adhesion of pathogens [4,5].Fucoidan is classically known as a selectin and scavenger receptor blocking agent [6].By blocking these cellular adhesion molecules, fucoidan can prevent the intrusion of neu-trophils into tissue spaces, attenuating inflammatory responses. Pharmaceutical agents thattarget a specific pro-inflammatory pathway in AD have been greatly explored [7]. Fucoidanhas significantly reduced the elevated level of IgE in human peripheral blood mononuclearcells of AD patients in vitro [8] and topical fucoidan has been shown to ameliorate AD in amouse model [9,10]. Despite these beneficial effects of fucoidan in AD, it is not known howfucoidan affects human epidermal keratinocytes and its gene expression.

Reconstructed human epidermis (RHE) provides a great in vitro research platform, sinceit mimics layering, differentiation and barrier function of normal human in vivo epidermis [11].Keratinocytes form the outermost layer of the skin and constitute 90% of epidermal skin

Dermatopathology 2021, 8, 69–83. https://doi.org/10.3390/dermatopathology8020012 https://www.mdpi.com/journal/dermatopathology

Dermatopathology 2021, 8 70

cells. When pathogens are introduced onto the surface of skin, keratinocytes begin to producepro-inflammatory mediators, leading to phenotype features of AD [12]. AD is characterizedby an imbalance of the Th1/Th2 and during the acute phase of the disease, Th2-mediatedcytokines such as interleukin-4, -13 and-25 are overly expressed [13]. Introduction tothese interleukins to RHE induces histological and gene expressions changes typical to theAD patients [14].

In this research, we sought to understand how fucoidan might affect gene expression andperiostin production in an in vitro 3D-RHE of keratinocyte model of atopic dermatitis usingTh2-mediated cytokines. Periostin is significantly overexpressed in AD patients [15–18] and inour Th2 cytokine stimulated model [14,19]. We then explored how fucoidan could affectthe adhesion of three types of human skin bacteria—the pathogenic Staphylococcus aureus,commensal Staphylococcus epidermidis and Cutibacterium acne.

2. Methods and Materials2.1. Materials

Fucoidan extracts from Fucus versiculosus (FV) and Undaria pinnatifida (UP) wereprovided by Marinova Pty Ltd. (Cambridge, Australia). The proprietary aqueous extractwas designed for topical application and specific properties are described in Table 1.

Table 1. Description of Fucus versiculosus (FV) and Undaria pinnatifida (UP).

FucoidanExtract

NeutralCarbohydrates Sulfate Cations

(approx.) Fucoidan Polyphenol

FV 43.7% 10.1% 3% 58.6% 33.7%UP 48.8% 27.4% 9% 89.6% <2%

2.2. Reconstructed Human Epidermis—RHE

The study was carried out on epidermis in vitro, reconstituted with NHEKs ker-atinocytes isolated from foreskin of 3 Caucasian donors (RHE). The tissues were culturedat the air–liquid interface, during 14 days, in Epilife medium (Fisher Scientific, Merelbeke,Belgium, M-EPI-500-A) containing supplements and antibiotics (Gentamycin, Fisher Scien-tific, 15710-049). They were maintained in a humid atmosphere at 37 ◦C with 5% CO2.

2.3. Induction of the Th2-Inflamed Model

Three Th2-related cytokines (IL-4, IL-13 and IL-25) interleukins were applied onthe culture medium of RHE at the concentrations of 50 ng/mL for IL-4 and IL-13, and20 ng/mL for IL-25, during 48 h to induce alterations and gene expression modulationsreminiscent to AD and a sensitive skin [14,20–22].

2.4. Treatment of the Epidermis—Gene Expression

FV and UP were diluted in PBS at 100 µg/mL and applied topically on RHE duringthe 48 h of stimulation. A mesh has been applied on the stratum corneum to allow the evendistribution of testing compounds on the tissue surface. For control PBS had been appliedtopically on Th2-stimulated RHE, with addition of a mesh.

As a reference treatment GW3965 (10 µM) was applied in the culture medium duringthe same 48 h in combination with the cytokines. GW3965 is an LXR agonist that modulatesthe expression of genes involved in lipid homeostasis and inflammation with therapeuticpotential in AD [14,23,24]. This condition was compared to the corresponding controlcondition, treated only with DMSO at 0.05%, given the DMSO was used for the preparationof GW3965 in the culture medium.

2.5. Analysis of Tissue Morphology

The tissue morphology was analyzed through a haematoxylin/eosin (H/E) staining.At the end of 48 h treatment, the tissues were fixed in 4% formaldehyde solution, dehydrated

Dermatopathology 2021, 8 71

and embedded in paraffin. Sections of 6 µm thick of tissue were generated using Leica micro-tome RM2245 and laid over microscopic slides before staining with H/E used to visualizegeneral tissue morphology. Slides were mounted with specific medium and examined with aLeica DM2000 photomicroscope coupled to a digital camera (Zeiss DFC420C).

2.6. Analysis of Gene Expression Modifications2.6.1. Total RNA Extraction and Integrity Analysis

At the end of the treatment, total RNAs were extracted using the Qiagen RNeasy kit(Qiagen; 74106). RHE were rinsed with cold PBS and lysed in the ad hoc buffer from the kit.Extraction was performed according to the Manufacturer’s instructions. The integrity ofRNA was analyzed and described in Supplementary information (Figure S1). The collectedRNAs were stored at −80 ◦C.

2.6.2. cDNA Synthesis

Reverse transcription was performed with the high capacity RNA-to-cDNA kit(Applied Biosystems, Aalst, Belgium; 438706) from total RNA and the cDNAs were thenstored at −20 ◦C until use in polymerase chain reactions.

2.6.3. TaqMan Assays

The microfluidic TaqMan qPCR arrays were designed by StratiCELL and manufac-tured on demand by Applied Biosystems. Data on 96 genes of interest including 3 internalcontrols Carbonic anhydrase (CA2), Involucrin (IVL) and Loricirin (LOR), and 1 house-keeping gene for normalization purpose, B2M (β2-microglobuline), were gathered from theRHE samples under different conditions (Figure S2). The TaqMan arrays were processedas described by the manufacturer’s instructions (Micro Fluidic Card Getting Started Guide,Applied Biosystems).

Briefly, 4 µL (4 ng) of cDNA were mixed with 10 µL of TaqMan Fast Advanced MasterMix (Applied Biosystems), 1 µL of TaqMan Gene Expression Assay and 5 µL of RNAse freewater before being injected into the arrays and dispersed into the wells by centrifugation.Arrays were sealed and qPCRs were run using the Quantstudio7 Real-Time PCR System(Applied Biosystems) and its software (QuantStudio real time PCR Software v1.3., AppliedBiosystems). The thermal cycles were programmed with one first denaturation step at95 ◦C for 20 s. The amplification protocol was followed with 40 cycles (1 s at 95 ◦C and 20 sat 60 ◦C).

All the measurements were performed from triplicates of culture (n = 3) and thresholdcycles (CT) were obtained for each gene. The relative expression levels were calculated bythe comparative CT (∆∆CT) method through a combination of statistical analysis [25,26].Briefly, CT obtained from treated condition was compared with CT obtained the referencecondition. This average CT has been normalized to CT of B2M, the reference gene. Therelative quantification (RQ) is obtained using the formula below:

RQ = 2−(∆CT

treated condition − ∆CT

reference condition)

where ∆CT = CT[target gene] − CT[reference gene] in cDNA sample.The genes for which the CT values were >36 have been considered as non-expressed

and omitted of the analysis. These include CNR1, IL6, LCE3B and SEMA3A.In order to normalize the results, B2M was amplified from the same cDNA samples.

A control without cDNA was performed in parallel as negative control of amplification toverify the absence of contaminants.

The data obtained for the condition treated with FV and UP were compared to theuntreated condition stimulated by Th-2 cytokines. The maximum CT cut-off value wasfixed at 36 cycles.

Dermatopathology 2021, 8 72

2.7. Analysis of Extracellular Periostin Abundance

The quantifications of the extracellular releases of Periostin (Thermo Scientific,EHPOSTN) were performed on the supernatants of all conditions in a specific Elisa assay.

2.8. Bacterial Cultures, Adhesion of Bacteria, and CFU Counting2.8.1. Bacterial Cultures of Staphylococcus epidermis and Staphylococcus aureus

Staphylococcus aureus (ATCC 6538) and Staphylococcus epidermidis (ATCC 12228) wereobtained from the American Type Culture Collection. The strains were grown in TSB(Tryptic Soy Broth) at 37 ◦C under 150 rpm and on TSA (Tryptic Soy Agar) plate.

The overnight (O/N) cultures of S. epidermidis and S. aureus were prepared in 10 mLof TSB medium from a fresh colony obtained by culturing S. epidermidis and S. aureus onTSA agar. The resulting cultures were then centrifuged for 10 min at 7000× g to harvestthe cells and the pellets were re-suspended in 10 mL of PBS. The suspensions were thendiluted 1000 fold in order to obtain an inoculum of approximately 105 CFU/mL.

2.8.2. Adhesion of Bacteria and CFU Counting

Adhesion of bacteria has been assessed by quantification of colonies on microbiologicalplates (Colony Forming Unit counting). After 14 days of culture at the air-liquid interface,RHE surfaces were put into contact for 30 min with 100 µL of FV and UP at 100 µg/mLor 100 µL of PBS for control samples. After 30 min, 100 µL of the bacterial suspensionswere added on the RHE surface at a concentration of 105 CFU/mL for 1 h. Control sampleswere treated with 100 µL PBS. After the contact period, non-adherent cells were eliminatedby six consecutive rinses with 300 µL of sterile PBS. Finally, the adherent bacteria werecollected in PBS with 0.1% Tween80 with a swab. Both adherent and non-adherent cellswere enumerated by seeding on TS agar (according to the decimal dilution method).

3. Results3.1. Effects of the Test Compounds on the Morphology of RHE When Cultured under ConditionsMimicking Atopic Dermatitis

Morphology of epidermis was examined after 48 h of topical treatment of FV and UPby H/E staining. This was compared to RHE in the presence and absence of the GW3965for 48 h (Figure 1). RHE controls (Figure 1a) presented a full differentiation of keratinocytesfrom basal to cornified layers resulting in the formation of the four typical layers of theepidermis, namely, basal, spinous, granular and cornified layers.

The epidermal incubation with the mix of IL-4, IL-13 and IL-25 influenced the tissueorganization, indicating the tissue response to the inflamed environment (Figure 1b,c). This wasespecially observed in the deepest layers of the RHE and more so, the basal layer, with a loss ofpolarized orientation of the apical–basal axis and reduced cohesion. As expected, compared toPBS (Figure 1c) DMSO (Figure 1b) did not influence any structural changes to Th2 stimulatedtissue organization.

When the reference compound GW3965 was applied to the RHE, the morphologicalchanges induced by the Th2 stimulation were considerably reduced, prohibiting the alterationof the polarized orientation of the basal layer (Figure 1d). As expected, DMSO alone did notchange the morphology of the Th2–induced RHE, indicating that the GW3965 compound itselfis responsible for reducing morphology change brought by cytokines (Figure 1c). The topicaltreatment with 100 µg/mL of FV and UP on Th2-induced RHE did not lead to additionalmorphological changes however no cytotoxicity was observed (Figure 1e,f). This supports thenon-noxious impact of FV and UP on reconstructed epidermis.

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(a) (b)

(c) (d)

(e) (f)

Figure 1. Histological sections of Th2-stimulated reconstructed human epidermis (RHE) after 48 h or topical and systemic

treatments with the test compounds and reference of its solvent, respectively (H/E staining; n = 1). (a) control; (b) Th2 (+PBS

topic); (c) Th2 + DMSO 0.05% (d) Th2 + 10 µM GW3965 in 0.05% DMSO; (e) Th2 + 100 µ/mL FV; (f) Th2 + 100 µ/mL UP.

The epidermal incubation with the mix of IL-4, IL-13 and IL-25 influenced the tissue

organization, indicating the tissue response to the inflamed environment (Figure 1b,c). This

was especially observed in the deepest layers of the RHE and more so, the basal layer,

with a loss of polarized orientation of the apical–basal axis and reduced cohesion. As ex-

pected, compared to PBS (Figure 1c) DMSO (Figure 1b) did not influence any structural

changes to Th2 stimulated tissue organization.

When the reference compound GW3965 was applied to the RHE, the morphological

changes induced by the Th2 stimulation were considerably reduced, prohibiting the alter-

ation of the polarized orientation of the basal layer (Figure 1d). As expected, DMSO alone

did not change the morphology of the Th2–induced RHE, indicating that the GW3965

compound itself is responsible for reducing morphology change brought by cytokines

(Figure 1c). The topical treatment with 100 μg/mL of FV and UP on Th2-induced RHE did

not lead to additional morphological changes however no cytotoxicity was observed (Figure

1e,f). This supports the non-noxious impact of FV and UP on reconstructed epidermis.

Figure 1. Histological sections of Th2-stimulated reconstructed human epidermis (RHE) after 48 h ortopical and systemic treatments with the test compounds and reference of its solvent, respectively(H/E staining; n = 1). (a) control; (b) Th2 (+PBS topic); (c) Th2 + DMSO 0.05% (d) Th2 + 10 µMGW3965 in 0.05% DMSO; (e) Th2 + 100 µ/mL FV; (f) Th2 + 100 µ/mL UP.

3.2. Gene Expression Analysis by RT-qPCR Using TaqMan Array

To better understand whether topical treatment of FV and UP alters any AD relatedgene expressions, we analyzed 93 target genes that are specifically designed to assimilatein vitro AD environment.

First, to validate that introduction of Th2 related cytokines (IL-4, IL-13, and IL-25)can stimulate atopic dermatitis environment, we looked at differentially expressed geneswith p-value < 0.005. A large number of differentially expressed genes in Th2-inducedRHE were observed compared to the normal RHE environment as expected (Table 2).A global analysis of the differentially regulated genes by Th2 stimulation was carried out andvolcano plot was used to highlight significant variations of gene expression (Figure 2a).

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Table 2. Differentially expressed genes that are shown significant changes. The increases are in greenand decreases in red.

Th2-Induced RHE vs. RHE

Genes Fold Change p Value Genes Fold Change p ValueABCA1 0.33 0.0035 AHR 1.67 0.0118CASP14 0.49 0.0177 CA2 6.38 0.0002CCL26 0.43 0.0378 CAPN14 134.80 0.0000CDHR1 0.33 0.0001 CCL2/MCP1 3.86 0.0062CERS3 0.59 0.0126 CCL27 2.49 0.0141CLDN8 0.24 0.0003 CCL5/RANTES 5.02 0.0018CNR1 0.51 0.0346 CH25H 4.34 0.0043

CXCL10 0.23 0.0061 CTSC 4.50 0.0023DGAT2 0.52 0.0081 DUOX1 2.39 0.0279EDN1 0.39 0.007 FZD10 7.23 0.0062FA2H 0.23 0.0162 IL13RA2 8.07 0.0012FASN 0.66 0.0498 IL2R 14.60 0.001FLG 0.53 0.0321 NELL2 3.99 0.0112

LCE4A 0.58 0.0283 POSTN 8.60 0.0013LPIN1 0.54 0.0454 TNC 13.13 0.0034OCLN 0.68 0.0477 TNFAIP6 465.19 0.0086

SEMA3A 0.51 0.0336SULT1E1 0.46 0.0431

TAC1 0.51 0.014TRPV1 0.38 0.0195

Th2 Induced RHE vs. FV Treated Th2 Induced RHE vs. UP Treated

Genes Fold Change p Value Genes Fold Change p ValueCD44 0.71 0.0183 ABCA1 1.38 0.0369

S100A6 0.51 0.0425 CCL26 1.80 0.0457UGCG 1.46 0.0444

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Dermatopathology 2020, 8 75

(a)

(b)

Figure 2. Cont.

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Dermatopathology 2020, 8 76

(c)

Figure 2. Volcano plots in analyzing differential gene expressions comparing between (a) control RHE and Th2 induced-

RHE, (b) Th2 induced-RHE and FV treated Th2 induced-RHE and (c) Th2 induced-RHE and UP treated Th2 induced-RHE.

Fold change boundary: 1 and p-value boundary: 0.05.

Both topical treatments of FV and UP to Th2 induced-RHE induced significant changes

in the expression of a few genes compared to the stimulation alone (Table 2 and Figure 2b,c).

In the presence of FV, CD44 (CD44 antigen, hyaluronate receptor) and S100A6 (S100 cal-

cium binding protein A6) were down regulated, whereas UGCG (Ceramide glucosyltransfer-

ase) was upregulated. There were many non-significant modifications and this concerns es-

pecially the regulation of genes involved in lipid homeostasis and barrier recovery (Table

3). The majority of them appear to be regulated in an opposite direction than what observed

in tissue cultured in presence of Th2 cytokines alone.

The treatment of Th2 stimulated epidermis with UP tended to reverse a greater num-

ber of Th2-related effects on gene expression than found in response to treatment with FV.

Indeed, a broader panel of Th2 gene targets were inversely regulated after UP application

compared to FV application (Table 3).

Figure 2. Volcano plots in analyzing differential gene expressions comparing between (a) control RHE and Th2 induced-RHE, (b) Th2induced-RHE and FV treated Th2 induced-RHE and (c) Th2 induced-RHE and UP treated Th2 induced-RHE. Fold change boundary: 1and p-value boundary: 0.05.

Four genes that prompted most fold changes were TNFAIP6 (tumor necrosis factor-inducible gene 6 protein), CAPN14 (Calpain-14), IL2RG (IL-2 receptor subunit gamma)and TNC (Tenascin-C). These genes are all up regulated in Th2-induced RHE and areinflammatory mediators. Moreover, NELL2 (neural epidermal growth factor-like 2) andCA2 (carbonic anhydrase 2) that are representative of atopic dermatitis and differentiatesfrom psoriasis were also upregulated.

Down-regulated genes are OCLN, FASN, UGCG and CES3 mainly involved in barrierrecovery and lipid homeostasis.

Both topical treatments of FV and UP to Th2 induced-RHE induced significant changesin the expression of a few genes compared to the stimulation alone (Table 2 and Figure 2b,c).In the presence of FV, CD44 (CD44 antigen, hyaluronate receptor) and S100A6 (S100 calciumbinding protein A6) were down regulated, whereas UGCG (Ceramide glucosyltransferase)was upregulated. There were many non-significant modifications and this concerns espe-cially the regulation of genes involved in lipid homeostasis and barrier recovery (Table 3).The majority of them appear to be regulated in an opposite direction than what observedin tissue cultured in presence of Th2 cytokines alone.

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Table 3. The list for genes involved in complementary regulations about lipid homeostasis and potential regulations onbarrier recovery. The increases and decreases are in green and red, respectively, and the significant p-values in bold.

Functions GenesTh2 vs. NT Th2 vs. Th2 + FV Th2 vs. Th2 + UP

Fold Change p Value Fold Change p Value Fold Change p ValueTriglyceride

synthesis GPAT3 0.6214 0.0861 1.3189 0.1494 1.3213 0.1769

Fatty acidsynthesis

ACACA 0.6756 0.1961 1.2663 0.4387 1.6682 0.1628FA2H 0.2267 0.0162 1.2280 0.4934 2.0985 0.3626

Cholesterolbiosynthesisand transport

ABCA1 0.3335 0.0035 1.3755 0.0369 1.2663 0.0646HMGCS1 0.6389 0.1370 0.8291 0.4831 1.8712 0.0777NR1H3 0.7706 0.4183 1.3721 0.1926 1.1837 0.3636

SULT1E1 0.4577 0.0431 0.9815 0.9404 1.7573 0.1530

Ceramidesynthesis

CERS3 0.5949 0.0126 1.0840 0.4628 1.5486 0.0728GBA 0.7193 0.4457 1.3629 0.4651 1.7666 0.2421

SMPD1 0.7417 0.1241 2.8455 0.3736 1.3497 0.1891UGCG 0.6619 0.0501 0.9075 0.4411 1.4580 0.0444

AM defense DEFB4A 0.3933 0.0691 1.3601 0.5485 1.2980 0.4851Epidermal

biology HAS3 5.4933 0.1958 0.4924 0.5206 0.7897 0.8425

Epidermaljunction

CLDN25 1.4695 0.7438 0.6323 0.6919 1.5768 0.7407OCLN 0.6770 0.0477 1.0514 0.7956 1.2521 0.1352TJP1 0.6468 0.1899 1.6713 0.3112 1.3790 0.5035

Cornifiedenvelope

precursors

CASP14 0.4896 0.0177 0.9058 0.5509 1.3626 0.2183LCE2A 0.5544 0.1728 1.6514 0.4677 1.5951 0.4212

SPRR2A 0.5200 0.2194 1.6174 0.4346 0.8558 0.7255ZNF750 0.8382 0.5563 0.9098 0.7737 1.5126 0.3437

The treatment of Th2 stimulated epidermis with UP tended to reverse a greater numberof Th2-related effects on gene expression than found in response to treatment with FV.Indeed, a broader panel of Th2 gene targets were inversely regulated after UP applicationcompared to FV application (Table 3).

3.3. Effects of the Test Compounds on Periostin Release by RHE When Cultured under ConditionsMimicking Atopic Dermatitis

To investigate whether the FV and UP treatment could modulate the protein expressionof periostin, a well-known maker of AD [15–18], the analysis was performed by a specificimmunoassay (ELISA) targeting periostin. The changes of periostin production are shownin Figure 3a,b.

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Dermatopathology 2020, 8 77

Table 3. The list for genes involved in complementary regulations about lipid homeostasis and potential regulations on

barrier recovery. The increases and decreases are in green and red, respectively, and the significant p-values in bold.

Functions Genes Th2 vs NT Th2 vs Th2 + FV Th2 vs Th2 + UP

Fold Change p Value Fold Change p Value Fold Change p Value

Triglyceride synthesis GPAT3 0.6214 0.0861 1.3189 0.1494 1.3213 0.1769

Fatty acid synthesis ACACA 0.6756 0.1961 1.2663 0.4387 1.6682 0.1628

FA2H 0.2267 0.0162 1.2280 0.4934 2.0985 0.3626

Cholesterol biosynthesis

and transport

ABCA1 0.3335 0.0035 1.3755 0.0369 1.2663 0.0646

HMGCS1 0.6389 0.1370 0.8291 0.4831 1.8712 0.0777

NR1H3 0.7706 0.4183 1.3721 0.1926 1.1837 0.3636

SULT1E1 0.4577 0.0431 0.9815 0.9404 1.7573 0.1530

Ceramide synthesis

CERS3 0.5949 0.0126 1.0840 0.4628 1.5486 0.0728

GBA 0.7193 0.4457 1.3629 0.4651 1.7666 0.2421

SMPD1 0.7417 0.1241 2.8455 0.3736 1.3497 0.1891

UGCG 0.6619 0.0501 0.9075 0.4411 1.4580 0.0444

AM defense DEFB4A 0.3933 0.0691 1.3601 0.5485 1.2980 0.4851

Epidermal biology HAS3 5.4933 0.1958 0.4924 0.5206 0.7897 0.8425

Epidermal junction

CLDN25 1.4695 0.7438 0.6323 0.6919 1.5768 0.7407

OCLN 0.6770 0.0477 1.0514 0.7956 1.2521 0.1352

TJP1 0.6468 0.1899 1.6713 0.3112 1.3790 0.5035

Cornified envelope precursors

CASP14 0.4896 0.0177 0.9058 0.5509 1.3626 0.2183

LCE2A 0.5544 0.1728 1.6514 0.4677 1.5951 0.4212

SPRR2A 0.5200 0.2194 1.6174 0.4346 0.8558 0.7255

ZNF750 0.8382 0.5563 0.9098 0.7737 1.5126 0.3437

3.3. Effects of the Test Compounds on Periostin Release by RHE When Cultured under

Conditions Mimicking Atopic Dermatitis

To investigate whether the FV and UP treatment could modulate the protein expression

of periostin, a well-known maker of AD [15–18], the analysis was performed by a specific

immunoassay (ELISA) targeting periostin. The changes of periostin production are shown

in Figure 3a,b.

(a) (b)

Figure 3. Changes in the periostin (POSTN) abundance in (a) Th2-induced RHE in 0.05% DMSO and in the presence of GW3965

and (b) in the presence of FV and UP. Data are given in percentage relative to the untreated condition. (*** p < 0.001 and ** 0.001

< p < 0.01).

The Th2 stimulation (+DMSO) induced a strong and significant production of peri-

ostin as compared to unstimulated condition (Figure 3a). As expected, the presence of

GW3965 allowed to significantly decrease this induction, favoring a return towards a ba-

sal level of protein expression [14].

0

200

400

600

800

1000

1200

1400

PO

ST

N (

% r

elat

ive

to R

HE

)

RHE Th2 + DMSO Th2 + GW3965

***

***0

200

400

600

800

1000

1200

PO

ST

N (

% r

elat

ive

to R

HE

)

RHE Th2 Th2

+ FV

Th2

+ UP

**

nsns

Figure 3. Changes in the periostin (POSTN) abundance in (a) Th2-induced RHE in 0.05% DMSO and in the presence ofGW3965 and (b) in the presence of FV and UP. Data are given in percentage relative to the untreated condition. (*** p < 0.001and ** 0.001 < p < 0.01).

The Th2 stimulation (+DMSO) induced a strong and significant production of periostinas compared to unstimulated condition (Figure 3a). As expected, the presence of GW3965allowed to significantly decrease this induction, favoring a return towards a basal level ofprotein expression [14].

The Th2 challenge combined with topical application of PBS triggered an importantand significant release of periostin. It is interesting to note that the induction level ofperiostin protein expression was found similar to those of its gene expression, supportingthe relevance to study this marker in the present model. In a similar way, the addition ofFV or UP stimulated a reduced production of periostin shown in Figure 3b. Although notsignificant, this downward tendency supports a protective effect of FV and UP treatmentson the regulation of periostin.

3.4. Adhesion of Bacteria

Staphylococcus aureus colonizes the skin of majority of atopic dermatitis patients andit could lead to increase disease severity. To test whether fucoidan could prevent orminimize bacteria adhesion, we evaluated the impact of FV and UP at 100 µg/mL on theadhesion of Staphylococcus epidermidis, Staphylococcus aureus and Cutibacterium acnes on RHE.Adhesion of bacteria has been assessed by quantification of colonies on microbiologicalplates after collection of adherent and non-adherent cells from the RHE tissues, as explainedin the methods.

There was no significant effect neither with FV or UP on the adhesion of the commensalS. epidermidis (Figure 4a). However, a significant reduction of S. aureus adhesion wasobserved upon prior topical application of FV and UP on the stratum corneum of RHE(Figure 4b). Furthermore, topical application of FV and UP reduces the adhesion of the C.acnes (Figure 4c).

Dermatopathology 2021, 8 79

Dermatopathology 2020, 8 78

The Th2 challenge combined with topical application of PBS triggered an important

and significant release of periostin. It is interesting to note that the induction level of per-

iostin protein expression was found similar to those of its gene expression, supporting the

relevance to study this marker in the present model. In a similar way, the addition of FV

or UP stimulated a reduced production of periostin shown in Figure 3b. Although not

significant, this downward tendency supports a protective effect of FV and UP treatments

on the regulation of periostin.

3.4. Adhesion of Bacteria

Staphylococcus aureus colonizes the skin of majority of atopic dermatitis patients and

it could lead to increase disease severity. To test whether fucoidan could prevent or min-

imize bacteria adhesion, we evaluated the impact of FV and UP at 100 μg/mL on the ad-

hesion of Staphylococcus epidermidis, Staphylococcus aureus and Cutibacterium acnes on RHE.

Adhesion of bacteria has been assessed by quantification of colonies on microbiological

plates after collection of adherent and non-adherent cells from the RHE tissues, as ex-

plained in the methods.

There was no significant effect neither with FV or UP on the adhesion of the com-

mensal S. epidermidis (Figure 4a). However, a significant reduction of S. aureus adhesion

was observed upon prior topical application of FV and UP on the stratum corneum of RHE

(Figure 4b). Furthermore, topical application of FV and UP reduces the adhesion of the C. acnes

(Figure 4c).

(a) (b)

(c)

Figure 4. Concentrations of adherent (a) S. epidermidis, (b) S. aureus and (c) C. acnes after topical treatment with FV and

UP. The concentrations are expressed in CFU/RHE. The graph represents the means obtained on three independent cul-

tures with three counts for each of them, as well as the standard deviation. The values of 0.001 < p < 0.05 are considered as

significant (*). Student’s test compared to control.

0.00E+00

5.00E+03

1.00E+04

1.50E+04

2.00E+04

Ad

he

ren

t ce

lls (

CFU

/RH

E)

Control FV UP

2.0 x 104

1.5 x 104

1.0 x 104

0.5 x 104

0 0.00E+00

2.00E+03

4.00E+03

6.00E+03

8.00E+03

1.00E+04

1.20E+04

Ad

he

ren

t ce

lls (

CFU

/RH

E)

Control FV UP

**

1.2 x 104

0.8 x 104

0.4 x 104

0

0.00E+00

1.00E+04

2.00E+04

3.00E+04

4.00E+04

5.00E+04

6.00E+04

7.00E+04

Ad

he

ren

t ce

lls (

CFU

/RH

E)

Control FV UP

**

0

2.0 x 104

4.0 x 104

6.0 x 104

Figure 4. Concentrations of adherent (a) S. epidermidis, (b) S. aureus and (c) C. acnes after topical treatment with FV and UP.The concentrations are expressed in CFU/RHE. The graph represents the means obtained on three independent cultureswith three counts for each of them, as well as the standard deviation. The values of 0.001 < p < 0.05 are considered assignificant (*). Student’s test compared to control.

4. Discussion

Sensitive skin with atopic tendency and atopic eczema are common skin problems/diseasesassociated with altered epidermal barrier and, for the most severe cases, chronic inflam-mation. Atopic dermatitis has a strong Th2 component associated with IL-4 and IL-13over-production by Th2-polarized lymphocytes in the acute phase of the disease.

The RHE model mimicking an inflammatory context through Th2 cytokines stimula-tion and used in this study was fully characterized by the demonstration of the dysregula-tion of many genes involved in skin barrier, lipid metabolism and transport, axon guidanceand inflammation. These data are in correlation with other published studies on closelyrelated in vitro models [14,22,27,28].

Both treatments of Th2-induced RHE with FV and UP stimulated significant changesin expression of a few genes compared to the Th2 induction alone. Insoluble lipids requirespecific transport mechanisms or carriers such as ABCA1 to move them through thecytoplasm [29]. Keratinocytes require abundant cholesterol for cutaneous permeabilitybarrier function, hence the regulation of cholesterol homeostasis is of great importance.ABCA1 is a ubiquitous membrane transporter responsible for cholesterol efflux and playspivotal role in regulating cellular cholesterol levels. A significant decrease of the ABCA1expression following Th2 stimulation was restored and increased its expression by 1.38fold by FV. Therefore, FV considerably improve or stabilize the cholesterol homeostasis,through the up-regulation of ABCA1, in tissues whose barrier function is impaired incontext of Th2 inflammation.

Dermatopathology 2021, 8 80

Interestingly, FV also induced the significant gene regulations of CCL26, a chemokineinvolved in the recruitment of inflammatory cells. The increased expression of the Th2-down-regulated chemokine may support the ability of FV to reverse the Th2 effect, but alsopromote the recruitment of cells involved in wound healing process [30,31].

The differential expression of CD44, the receptor for hyaluronic acid, was also reversedin the presence of UP. Increased expression of the hyaluronic synthase 3 (HAS3) by Th2stimulation was also decreased in response to FV application. These co-regulated genesmay indicate the ability of fucoidan to act on the metabolism of hyaluronic acid and relatedpathways, and more so, to counteract spongiosis, a hallmark of eczema [32]. In spongiosis,keratinocytes lose cohesiveness with a decreased expression of cadherins, as a water influxinto the epidermal intercellular spaces occurs together with an accumulation of hyaluronate(HA). This phenomenon is associated with an increased expression of CD44, the majorreceptor of HA and HAS3. All these components are known to be up-regulated in lesionalAD skin as a consequence to Th2 inflammation [32–35].

S100A6 encodes for a calcium binding protein of the S100 family and is implied in theregulations of the differentiation process of keratinocyte [36] and was down-regulated byUP. The overexpression of this gene of the S100 family in keratinocyte is associated with anundifferentiated status of the cell. Therefore, the great down-regulation of S100A6 by thetest compound could demonstrate a promoted differentiation of keratinocyte.

UGCG participates in the glycosylation of ceramides. One of remarkable and epidermis-specific ceramide species is acylceramides and they are essential for skin barrier formation.Decreases in acylceramide levels as well as alteration in ceramide composition and chain-lengthare associated with cutaneous disorders such as ichthyosis and psoriasis, and represented ahallmark of AD [37,38]. Upregulation of UGCG by UP could, therefore, have a positive effecton ceramides metabolism in the skin, by a return to normal level and on the restoration of theresulting barrier function in the event of an inflammatory challenge.

A large number of subtle modifications were also observed (Table 3). The majority ofaffected genes are involved in lipid homeostasis and barrier recovery and are regulatedin an opposite way compared to Th2-induced RHE. Although not significant, all thesesubtle but concomitant regulations might demonstrate a bundle of evidence which actsin complementarity to generate epidermal benefits against AD-related detrimental effects.Therefore, fucoidan could act in favor of a reinforced and more resistant epidermal barrier,alleviating the damages associated with sensitive skin and AD.

Most importantly fucoidan significantly inhibited adhesion of S. aureus which istypically abundant in skins of AD patients and aggravate the condition [39]. However,interestingly fucoidan did not affect the adhesion of S. epidermidis, usually innocuouscommensal skin bacteria. It is unclear how fucoidan differentiates between S. aureus andS. epidermidis. As a relatively hydrophobic bacteria, S. epidermis was found to adhere tostainless steel more effectively than to reconstituted skin. However, the opposite was truefor S. aureus which is known to have a basic and hydrophilic surface [40]. We speculatethat negatively charged fucoidan binds to S. aureus thereby preventing it to adhere to theRHE in this study.

Interestingly, antimicrobial activity against E. coli and S. aureus of fucoidan has beenobserved before [41] and showed a great potential as an antiadhesive biomaterial coatingagent that stops infection of S. aureus for urinary catheter applications [42].

Acne is a common skin disease which affects 9.4% of the global population [43] andover-colonization of Cutibacterium acnes is one of the main triggers for acne. Interestingly,recent studies indicated that the balance of C. acnes and S. epidermidis population on skin isvery important as S. epidermidis can limit C. acnes over-colonization [44]. Considerable inhi-bition of adhesion of C. acnes and maintenance of S. epidermidis population may potentiatefucoidan as a beneficial therapeutic agent in acne treatment.

Dermatopathology 2021, 8 81

5. Conclusions

Topical application of fucoidan from Fucus versiculosus and Undaria pinnatifida leads tonon-cytotoxic impact on the 3D reconstructed human epidermis. Together with beneficialgene regulation, subtle inhibition of periostin release and remarkable inhibition of S. aureusadhesion, fucoidan offers a promising therapeutic potential against skin inflammatorydisease such as atopic dermatitis and related damages.

Supplementary Materials: The following are available online at https://www.mdpi.com/2296-3529/8/2/12/s1, Figure S1: Analysis of the integrity of extracted RNA through their electrophoresisprofiles, Figure S2: Modifications of the gene expression of 3 targets from reconstructed epidermisafter 48 h of treatment with Th2-like cytokines in presence or absence of GW3965.

Author Contributions: Conceptualization, J.H.F., R.H., M.S.; methodology, R.H., M.S., A.C.; formalanalysis, C.L., M.B., A.C.; investigation, C.L., M.B.; writing—original draft preparation, A.Y.P., J.H.F.;writing—review and editing, A.Y.P., J.H.F., R.H.; visualization, C.L., M.B., A.C.; All authors have readand agreed to the published version of the manuscript.

Funding: This research was funded by Marinova Pty Ltd., located in Cambridge, Tasmania, Australia.

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Conflicts of Interest: Ah Young Park and Janet Helen Fitton are employees of Marinova Pty Ltd.Maureen Bourtembourg, Aline Chrétien, Roland Hubaux, Céline Lancelot and Michel Salmon areemployees of StratiCELL.

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